Myosin is a family of proteins that demonstrates force-generating ATPase activity when it interacts with actin. Myosin heavy chain (MHC) protein isoform diversity in vertebrates is generated by multiple genes as well as by alternative splicing of the pre-mRNA transcript. A previous study in this laboratory demonstrated the existence of neuron-specific isoforms of nonmuscle MHC-B, which may be generated by alternative splicing of pre-mRNA (Takahashi et al., J. Biol. Chem. 267, 1764, 1992). To prove and study the neuron-specific alternative splicing mechanism(s), we have isolated human genomic DNA clones which encode this region of nonmuscle MHC-B. Exons 5 and 6 (tentative), which encode part of the ATP binding site, are spliced constitutively and the distance between these two exons is approximately 8 kb. Between these two exons, two alternative exons, one of 30 nts and a second one of 18 nts 3' to the 30 nt exon, have been identified. The 30 nt exon is followed by the unconventional intron donor sequence, GCAAGT. The 30 nt exon sequence also corresponds to the neuron-specific inserted sequence in the cDNA which has been previously cloned. This exon alone or along with the 18 nt exon can be spliced into the mRNA from cultured human retinoblastoma cells (Y79 cells; Itoh and Adelstein, J. Biol. Chem. 270, 14533, 1995). In an attempt to localize the critical region of the pre-mRNA that is required for regulated alternative splicing, we have constructed a minigene with deletions and/or mutations using a eukaryotic expression vector. Following transfection into various cultured cells, including Y79 cells, the mRNAs derived from the minigene constructs were analyzed by RT-PCR. Deletion and mutation analysis demonstrated that a 20 nt sequence, located approximately 1.5 kb downstream from the 30 nt alternative exon is required for neuron- specific splicing.